Increased activity of the polyol pathway has been shown to play a role in diabetic complications of the nerve, kidney, and ocular tissues. Our studies have been involved in characterizing the structure and function of the two enzymes of the polyol pathway, aldose reductase (AR) and sorbitol dehydrogenase (SDH). Using site-directed mutagenesis, we defined specific amino acid residues of AR that are critical for the catalytic mechanism and inhibition of AR. For example, mutations at positions tryptophan 20 and tryptophan 111 of AR decreased the efficacy of AR inhibitors by more than 100 fold. We have also characterized the AR gene and its regulatory elements. Constructs of AR promoter fragments linked to the luciferase reporter gene defined the cis-element responsible for increased AR promoter activity under hyperosmotic stress. By using electrophoretic mobility shift assays and in vivo exonuclease III mediated footprinting, a putative transcription binding factor was localized to a 200 bp cis-element involved in this upregulation. Studies on the enzyme sorbitol dehydrogenase have included characterizing the cis-elements involved in SDH basal promoter activity. A negative regulatory element was found at approximately -2 Kb from the transcription initiation start site. SDH promoter/luciferase reporter gene expression was not observed to increase in cells osmotically stressed. These studies have focused on the mechanisms that control the action and regulation of the two enzymes of the polyol pathway. This information is pertinent to the problem of controlling overexpression of this pathway in diabetic patients.